Project Summary Ribosome and proteomic profiling have revealed a large number of small translated open reading frames (ORF) within previously described ?untranslated regions? (UTRs) and long non-coding RNAs. While some of the small ORFs depend on the encoded peptide to function in various fundamental processes (e.g., development). Translation of small ORFs in the 5?UTR, known as upstream-ORFs (uORFs), usually represses gene expression, independent of the encoded peptide. Small ORFs have also been reported in 3'UTR, termed downstream ORF (dORF). However, the dORF function and their relationship to human health and disease remain unknown. I characterized dORFs from human and zebrafish using ribosome profiling data. My preliminary data indicates, contrary to uORFs, translation of dORFs (small ORF in the 3?UTR) strongly enhances translation of the canonical ORFs and remains an uncharacterized regulatory mechanism across vertebrates. The objectives are: 1) Dissect at the single molecular level how dORF enhances translation of the canonical CDS. And 2) Determine whether alternative polyadenylation in cancer influences dORF regulation to cause cancer. The rationale for the proposed research is to gain a mechanistic understanding of dORF-mediated regulation and to assess the possible biological importance of dORF dysregulation under disease conditions (e.g. Cancer). This proposal is conceptually innovative as it is based on the exploration of a novel, yet widespread translation regulatory mechanism conserved across vertebrates. Technically, this proposal will combine single molecular imaging, genomic profiles (RNA-seq, Ribosome profiling), and reporter approaches in different human cell lines (including cancer cells) and published patient data. The outcomes from this project will emphasize the role of ribosome as a master gene expression regulator, and shield light on the importance of small ORFs. This translation kinetics work about dORF will provide critical insights into the molecular mechanism of this uncharacterized regulatory pathway. Exploring dORF dysregulation in cancer due to APA will highlight the mRNA itself as disease driver even without any mutation in DNA, and it also indicates possible clinical impact of dORF to detect and even cure cancer. My long-term interest is to study gene expression dysregulation in cancer. This training award will increase my knowledge background of cancer biology, molecular biology and bioinformatics. It will also promote the technical training of single molecular imaging, ribosome profiling, cell biology assays for cancer. Overall, this proposal will help me for future independent cancer molecular/genomic career.